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| United States Patent |
6,124,106
|
|
Wallace
, et al.
|
September 26, 2000
|
Method for detecting cancers
Abstract
The invention provides for the production of several humanized murine
antibodies specific for the antigen LK26, which is recognized by the
murine antibody LK26. This antigen is expressed in all choriocarcinoma,
teratocarcinoma and renal cancer cell lines whereas it is not expressed on
cell lines of leukaemias, lymphomas, neuroectodermally-derived and
epithelial tumour cell lines (excepting a small subset of epithelial cell
lines). Furthermore, whereas renal cancer cell lines express the LK26
antigen, normal renal epithelial cells do not. Similarly, with the
exception of the trophoblast, all normal adult and fetal tissues tested
are negative for the LK26 phenotype. The invention also provides for
numerous polynucleotide encoding humanized LK26 specific antibodies,
expression vectors for producing humanized LK26 specific antibodies, and
host cells for the recombinant production of the humanized antibodies. The
invention also provides methods for detecting cancerous cells (in vitro
and in vivo) using humanized LK26 specific antibodies. Additionally, the
invention provides methods of treating cancer using LK26 specific
antibodies.
| Inventors:
|
Wallace; Thomas Paul (Gight, GB);
Harris; William Joseph (Carnoustie, GB);
Carr; Francis Joseph (Balmedie, GB);
Rettig; Wolfgang J. (Biberach, DE);
Garin-Chesa; Pilar (Biberach, DE);
Old; Lloyd J. (New York, NY)
|
| Assignee:
|
Ludwig Institute for Cancer Research (New York, NY)
|
| Appl. No.:
|
266119 |
| Filed:
|
March 10, 1999 |
| Current U.S. Class: |
435/7.23; 435/7.1 |
| Intern'l Class: |
G01N 033/574 |
| Field of Search: |
530/387.3
435/4,7.1,7.21,7.23,7.9
|
References Cited
U.S. Patent Documents
| 4851332 | Jul., 1989 | Rettig et al. | 435/7.
|
| 5585089 | Dec., 1996 | Queen et al. | 424/133.
|
| 5646253 | Jul., 1997 | Wallace et al.
| |
| Foreign Patent Documents |
| 0197435 | Mar., 1986 | EP.
| |
| 04381 | Mar., 1992 | WO.
| |
Other References
Kabat et al, "Sequences of Proteins of Immunological Interest" 4th Ed. pp.
41, 45, 164, 169, 171, 1987.
|
Primary Examiner: Huff; Sheela
Attorney, Agent or Firm: Fulbright & Jaworski, LLP
Parent Case Text
This application is a divisional of application Ser. No. 08/760,840, filed
Dec. 5, 1996, now U.S. Pat. No. 5,952,484, which is a divisional of
application Ser. No. 08/207,996, filed Mar. 8, 1994, now U.S. Pat. No.
5,646,253.
Claims
What is claimed is:
1. A method for detecting presence of cancer cells which present antigen
LK26 on their surfaces in a sample, comprising contacting said sample with
a humanized antibody specific for LK 26 antigen, said humanized antibody
comprising a variable region heavy chain and a variable region light
chain, wherein said variable region heavy chain comprises an amino acid
sequence selected from the group consisting of LK26HuVH (SEQ ID NO: 18),
LK26HuVHFAIS,N (SEQ ID NO: 19), LK26HuVHSLF (SEQ ID NO: 22), and said
variable region light chain comprises an amino acid sequence selected from
the group consisting of LK26HuVK (SEQ ID NO: 23), LK26HuVKPW,Y (SEQ ID NO:
25 with the proviso that amino acid 72 is not Phe but is Tyr), and
determining binding of said humanized antibody to LK 26 antigen as a
determination of cancer cells which present antigen LK26 on their surfaces
in said sample.
2. The method according to claim 1 wherein the humanized antibody comprises
the heavy chain variable legion LK26KOLHuVH (SEQ ID NO: 22) and the light
chain variable region LK26HuVKPW,Y (SEQ ID NO: 25 with the proviso that
amino acid 72 is not Phe but is Tyr).
3. The method according to claim 1 wherein the humanized antibody comprises
the heavy variable region LK26HuVHSLF (SEQ ID NO: 21) and the light chain
variable region LK26HuVKPW,Y (SEQ ID NO: 25 with the proviso that amino
acid 72 is not Phe but is Tyr).
4. The method according to claim 1 wherein the humanized antibody comprises
the heavy chain variable region LK26HuVHFAIS,N (SEQ ID NO: 19) and the
light chain variable region LK26HuVKPW,Y (SEQ ID NO: 25) with the proviso
that amino acid 72 is not Phe but is Tyr).
5. The method of claim 1, wherein said cancer cells are choriocarcinoma,
teratocarcinoma, or renal cancer cells.
6. The method of claim 1, wherein said humanized antibody is labelled with
a detectable label.
7. The method of claim 1, comprising detecting said cancer cells in vivo.
8. The method of claim 6, wherein said detectable label is radioactive,
flourescent, or chromophoric.
9. The method of claim 8, wherein said detectable label is .sup.131 I,
.sup.125 I, or .sup.14 C.
10. The method of claim 8, wherein said detectable label is fluorescein,
phycolipoprotein, or tetraethlcrhodamine.
11. The method of claim 8, wherein said detectable label is an enzyme.
12. The method of claim 6, wherein said detectable label has a visible
color.
13. The method of claim 6, wherein said detectable label is an
agglutinizable label.
14. The method of claim 6, wherein said detectable label is electron dense.
15. The method of claim 14, wherein said detectable label is ferritin,
peroxidase, or a gold bead.
Description
1. FIELD OF THE INVENTION
The present invention is related to the field of molecular biology, and
more particularly to humanized antibodies.
2. BACKGROUND
The present invention provides novel recombinant immunoglobulins specific
for the human LK26 cancer antigen, polynucleotides encoding the novel
immunoglobulins, and host cells containing the novel polynucleotides. The
invention also provides methods for the production of these recombinant
antibodies, for the diagnosis and treatment of certain human cancers.
Transformation of a normal cell to a malignant cell is often accompanied by
a change in the expression of cell surface antigens. These changes in the
cell surface can be detected using monoclonal antibodies specific for such
antigens. In this way, different cancer cells can be detected and
characterized (Lloyd, K. O. (1983) "Human Tumour Antigens: Detection and
Characterization with Monoclonal Antibodies" in R. B. Herberman, ed.,
Basic and Clinical Tumour Immunology, pp 159-214, Martinus Nijhoff,
Boston).
European Patent Application Number 86104170.5 (Rettig) describes the
generation and characterization of the murine monoclonal antibody `LK26`.
The antibody was generated by the application of the hybridoma technology
of Kohler and Milstein (Kohler, G. and Milstein, C. (1975) Nature
256:495-497). The antibody specifically recognizes a cell surface
glycoprotein of molecular weight between 30 and 35 kDa (the LK26 antigen).
This antigen is expressed on all choriocarcinoma, teratocarcinoma and
renal cancer cell lines whereas it is not expressed on cell lines of
leukaemias, lymphomas, neuroectodermally-derived and epithelial tumour
cell lines (excepting a small subset of epithelial cell lines).
Furthermore, whereas renal cancer cell lines express the LK26 antigen,
normal renal epithelial cells do not. Similarly, with the exception of the
trophoblast, all normal adult and fetal tissues tested are negative for
the LK26 phenotype.
The specificity of the LK26 murine antibody makes it a powerful tool for
the detection and characterization of particular human cancer types in
vitro. However, the in vivo use of murine antibodies as agents for the
diagnosis and treatment of human diseases is severely curtailed by a
number of factors. Specifically, the human body recognizes murine
antibodies as foreign. This can elicit a human anti-mouse antibody (HAMA)
response (Schroff, R. et al. (1985) Cancer Res. 45:879-885) which results
in rapid clearance of the antibody from the circulation. Furthermore, the
Fc portion of a murine antibody is not as efficacious as the human Fc at
stimulating human complement or cell-mediated cytotoxicity. Therefore, it
is desirable to circumvent these problems associated with the in vivo use
of murine antibodies in diagnosis and therapy.
EP120694 (Celltech) and EP125023 (Genentech) disclose the development of
`chimeric` antibodies using recombinant DNA methods. Such antibodies
comprise the variable regions from one species, e.g. mouse, and the
constant regions from another species, e.g. human. Such chimeric
antibodies have the advantage that they retain the specificity of the
murine antibody but can also stimulate human Fc dependent complement
fixation and cell-mediated cytotoxicity. However, the murine variable
regions can still elicit a HAMA response (Bruggemann, M. et al. (1989) J.
Exp. Med. 170:2153-2157) thereby limiting the value of chimeric antibodies
as diagnostic and therapeutic agents.
British Patent Application Number GB2188638A (Winter) discloses a process
whereby recombinant antibodies can be generated by substitution of only
the variable region CDRs of one antibody with those from another.
Typically, this `CDR-grafting` technology has been applied to the
generation of recombinant, pharmaceutical antibodies consisting of murine
CDRs, human variable region frameworks and human constant regions (e.g.
Riechmann, L. et al., (1988) Nature 332:323-327). Such `reshaped` or
`humanized` antibodies have less murine content than chimeric antibodies
and retain the human constant regions necessary for the stimulation of
human Fc dependent effector functions. Consequently, humanized antibodies
are less likely than chimeric antibodies to evoke a HAMA response when
administered to humans, their half-life in circulation should approach
that of natural human antibodies and their diagnostic and therapeutic
value is enhanced.
In practice, simply substituting murine CDRs for human CDRs is not
sufficient to generate efficacious humanized antibodies retaining the
specificity of the original murine antibody. There is an additional
requirement for the inclusion of a small number of critical murine
antibody residues in the human variable region. The identity of these
residues depends on the structure of both the original murine antibody and
the acceptor human antibody. British Patent Application Number 9019812.8
describes a method for identifying a minimal number of substitutions of
foreign residues sufficient to promote efficacious antigen binding.
The invention described herein provides a process for the inclusion of
residues into the humanized antibodies which facilitates the proper
association of the VH and VL domains and thereby antigen binding.
The present invention provides novel, humanized monoclonal antibodies
specific for the human LK26 cancer antigen and various antibody
derivatives comprising humanized variable regions that are specific for
LK26. This has been achieved by the conversion of the murine LK26
monoclonal antibody described in European Patent Application Number
86104170.5 to humanized antibodies by utilizing CDR-grafting technologies.
The invention also provides methods for the production of these humanized
antibodies to be used in the diagnosis (both in vivo and in vitro) and
treatment of certain human cancers. Prior to the work of the inventors, it
was not known that the LK26 antibody or any other non-human antibody
specific for the the LK26 antigen could be humanized so as to retain
useful binding specificity.
3. BRIEF DESCRIPTION OF THE FIGURES
FIGS. 1(A-B) shows the DNA sequence (SEQ ID NO: 26 and SEQ ID NO: 27) and
corresponding amino acid sequence (SEQ ID NO: 28) of the murine LK26 heavy
chain variable region (VH). The CDRs are boxed. Underlined nucleotides and
amino acid residues are derived from the oligonucleotide primers used. The
backslash mark is used to indiacte the result obtained with the consensus
primers.
FIGS. 2(A-B) shows the DNA sequence (SEQ ID NO: 29 and SEQ ID NO: 31) and
corresponding amino acid sequence (SEQ ID NO: 30) of the murine LK26 light
chain variable region (VK). The CDRs are boxed. Underlined nucleotides and
amino acid residues are derived from the oligonucleotide primers used.
FIG. 3 shows the vector pSVgpt, which is used for the expression of
chimeric or humanized heavy chains in mammalian cells.
FIG. 4 shows the vector pSVhyg for the expression of chimeric or humanized
light chains in mammalian cells.
FIGS. 5-12 provide graphical data of ELISA results measuring the binding of
different humanized LK26 antibodies.
4. SUMMARY OF THE INVENTION
One aspect of the invention is to provide humanized antibodies specific for
the LK26 antigen.
Another aspect of the invention is to provide polynucleotides encoding
humanized antibodies specific for the LK26 antigens. Various expression
vectors comprising polynucleotides encoding humanized LK26 antibodies
joined to promoter sequences are also provided. Similarly, another aspect
of the invention is host cells transformed with expression vectors for the
expression of humanized LK26 specific antibodies.
Another aspect of the invention is to provide humanized anti-LK26
antibodies that are labeled with a detectable label or a therapeutic
label.
Another aspect of the invention is to provide methods for treating and/or
diagnosing cancer by administering a composition comprising a humanized
LK26 specific antibody. One method of defecting cancer cells involves the
steps of administering a labeled antibody (detectable label) to a patient
and subsequently detecting where in the body the labeled antibody has
bound.
5. DETAILED DESCRIPTION OF THE SPECIFIC EMBODIMENTS
As used herein, the term "humanized" antibody refers to a molecule that has
its CDRs (complementarily determining regions) derived from a non-human
species immunoglobulin and the remainder of the antibody molecule derived
mainly from a human immunoglobulin. The term "antibody" as used herein,
unless indicated otherwise, is used broadly to refer to both antibody
molecules and a variety of antibody derived molecules. Such antibody
derived molecules comprise at least one variable region (either a heavy
chain of light chain variable region) and include molecules such as Fab
fragments, Fab' fragments, F(ab').sub.2 fragments, Fd fragments, Fabc
fragments, Fd fragments, Fabc fragments, Sc antibodies (single chain
antibodies), diabodies, individual antibody light chains, individual
antibody heavy chains, chimeric fusions between antibody chains and other
molecules, and the like.
The term "conventional molecular biology methods" refers to techniques for
manipulating polynucleotides that are well known to the person of ordinary
skill in the art of molecular biology. Examples of such well known
techniques can be found in Molecular Cloning: A Laboratory Manual 2nd
Edition, Sambrook et al, Cold Spring Harbor, N.Y. (1989). Examples of
conventional molecular biology techniques include, but are not limited to,
in vitro ligation, restriction endonuclease digestion, PCR, cellular
transformation, hybridization, electrophoresis, DNA sequencing, cell
culture, and the like.
The term "variable region" as used herein in reference to immunoglobulin
molecules has the ordinary meaning given to the term by the person of
ordinary skill in the act of immunology. Both antibody heavy chains and
antibody light chains may be divided into a "variable region" and a
"constant region". The point of division between a variable region and a
heavy region may readily be determined by the person of ordinary skill in
the art by reference to standard texts describing antibody structure,
e.g., Kabat et al "Sequences of Proteins of Immunological Interest: 5th
Edition" U.S. Department of Health and Human Services, U.S. Government
Printing Office (1991).
The present invention provides humanized antibody molecules specific for
LK26 antigen in which at least parts of the CDRs of the heavy and/or light
chain variable regions of a human antibody (the receptor antibody) have
been substituted by analogous parts of CDRs of a murine monoclonal
antibody and the humanized antibody can specifically bind to the same as
the LK26 antibody. In a preferred embodiment of the subject invention, the
CDR regions of the humanized LK26 specific antibody are derived from the
murine antibody LK26. Some of the the humanized antibodies described
herein contain some alterations of the acceptor antibody, i.e., human,
heavy and/or light chain variable domain framework regions that are
necessary for retaining binding specificity of the donor monoclonal
antibody. In other words, the framework region of some embodiments the
humanized antibodies described herein does not necessarily consist of the
precise amino acid sequence of the framework region of a natural occurring
human antibody variable region, but contains various substitutions that
improve the binding properties of a humanized antibody region that is
specific for the same target as the murine LK26 specific antibody. A
minimal number of substitutions are made to the framework region in order
to avoid large-scale introductions of non-human framework residues and to
ensure minimal immunogenicity of the humanized antibody in humans. The
donor monoclonal antibody of the present invention is the LK26 murine
antibody, which is specific for the human LK26 cancer antigen.
The humanized antibodies of the present invention include complete antibody
molecules having full length heavy and light chains, or any fragment
thereof, such as the Fab or (Fab').sub.2 fragments, a heavy chain and
light chain dimer, or any minimal fragment thereof such as a Fv, an SCA
(single chain antibody), and the like, specific for the LK26 antigen
molecule.
In addition to providing for humanized LK26 specific antibodies, the
subject invention provides for polynucleotides encoding humanized LK26
specific antibodies. The subject polynucleotides may have a wide variety
of sequences because of the degeneracy of the genetic code. A person of
ordinary skill in the art may readily change a given polynucleotide
sequence encoding a humanized LK26 specific antibody into a different
polynucleotide encoding the same humanized LK26 specific antibody
embodiment. The polynucleotide sequence encoding the antibody may be
varied to take into account factors affecting expression such as codon
frequency, RNA secondary structure, and the like.
The humanized antibodies of the subject invention may be produced by a
variety of methods useful for the production of polypeptides, e.g. in
vitro synthesis, recombinant DNA production, and the like. Preferably, the
humanized antibodies are produced by recombinant DNA technology.
The humanized LK26 specific antibodies of the invention may be produced
using recombinant immunoglobulin expression technology. The recombinant
production of immunoglobulin molecules, including humanized antibodies are
described in U.S. Pat. No. 4,816,397 (Boss et al), U.S. Pat. No. 4,816,567
(Cabilly et al) U.K. patent GB 2,188,638 (Winter et al), and U.K. patent
GB 2,209,757. Techniques for the recombinant expression of
immunoglobulins, including humanized immunoglobulins, can also be found,
among other places in Goeddel et al, Gene Expression Technology Methods in
Enzymology Vol. 185 Academic Press (1991), and Borreback, Antibody
Engineering, W. H. Freeman (1992). Additional information concerning the
generation, design, and expression of recombinant antibodies can be found
in Mayforth, Designing Antibodies, Academic Press, San Diego (1993).
The recombinant humanized anti-LK26 antibodies of the invention may be
produced by the following process or other recombinant protein expression
methods:
a. Constructing, by conventional molecular biology methods, an expression
vector comprising an operon that encodes an antibody heavy chain in which
the CDRs and a minimal portion of the variable region framework that are
required to retain donor antibody binding specificity are derived from a
non-human immunoglobulin, such as the murine LK26 monoclonal antibody, and
the remainder of the antibody is derived from a human immunoglobulin,
thereby producing a vector for the expression of a humanized antibody
heavy chain.
b. Constructing, by conventional molecular biology methods, an expression
vector comprising an operon that encodes an antibody light chain in which
the CDRs and a minimal portion of the variable region framework that are
required to retain donor antibody binding specificity are derived from a
non-human immunoglobulin, such as the murine LK26 monoclonal antibody, and
the remainder of the antibody is derived from a human immunoglobulin,
thereby producing a vector for the expression of humanized antibody light
chain.
c. Transferring the expression vectors to a host cell by conventional
molecular biology methods to produce a transfected host cell for the
expression of humanized anti-LK26 antibodies.
d. Culturing the transfected cell by conventional cell culture techniques
so as to produce humanized anti-LK26 antibodies.
Host cells may be cotransfected with two expression vectors of the
invention, the first vector containing an operon encoding a heavy chain
derived polypeptide and the second containing an operon encoding a light
chain derived polypeptide. The two vectors may contain different
selectable markers but, with the exception of the heavy and light chain
coding sequences, are preferably identical. This procedure provides for
equal expression of heavy and light chain polypeptides. Alternatively, a
single vector may be used which encodes both heavy and light chain
polypeptides. The coding sequences for the heavy and light chains may
comprise cDNA or genomic DNA or both.
The host cell used to express the recombinant antibody of the invention may
be either a bacterial cell such as Escherichia coli, or preferably a
eukaryotic cell. Preferably a mammalian cell such as a chinese hamster
ovary cell, may be used. The choice of expression vector is dependent upon
the choice of host cell, and may be selected so as to have the desired
expression and regulatory characteristics in the selected host cell.
The general methods for construction of the vector of the invention,
transfection of cells to produce the host cell of the invention, culture
of cells to produce the antibody of the invention are all conventional
molecular biology methods. Likewise, once produced, the recombinant
antibodies of the invention may be purified by standard procedures of the
art, including cross-flow filtration, ammonium sulphate precipitation,
affinity column chromatography, gel electrophoresis and the like.
The humanized LK26 specific antibodies of the present invention may be used
in conjunction with, or attached to other antibodies (or parts thereof)
such as human or humanized monoclonal antibodies. These other antibodies
may be reactive with other markers (epitopes) characteristic for the
disease against which the antibodies of the invention are directed or may
have different specificities chosen, for example, to recruit molecules or
cells of the human immune system to the diseased cells. The antibodies of
the invention (or parts thereof) may be administered with such antibodies
(or parts thereof) as separately administered compositions or as a single
composition with the two agents linked by conventional chemical or by
molecular biological methods. Additionally the diagnostic and therapeutic
value of the antibodies of the invention may be augmented by labelling the
humanized antibodies with labels that produce a detectable signal (either
in vitro or in vivo) or with a label having a therapeutic property. Some
labels, e.g. radionucleotides may produce a detectable signal and have a
therapeutic property. Examples of radionuclide labels include .sup.125 I,
.sup.131 I, .sup.14 C. Examples of other detectable labels include a
fluorescent chromophore such as fluorescein, phycobiliprotein or
tetraethyl rhodamine for fluorescence microscopy, an enzyme which produces
a fluorescent or colored product for detection by fluorescence,
absorbance, visible color or agglutination, which produces an electron
dense product for demonstration by electron microscopy; or an electron
dense molecule such as ferritin, peroxidase or gold beads for direct or
indirect electron microscopic visualization. Labels having therapeutic
properties include drugs for the treatment of cancer, such as methotrexate
and the like.
The subject invention also provides for a variety of methods for treating
and/or detecting cancer cells. These methods involve the administration to
of humanized LK26 specific antibodies, either labelled or unlabelled, to a
patient. One method of detecting cancer cells in a human involves the step
of administering a labeled humanized LK26 specific antibody (labelled with
a detectable label) to a human and subsequently detecting bound labeled
antibody by the presence of the label.
The recombinant antibodies of this invention may also be used for the
selection and/or isolation of human monoclonal antibodies, and the design
and synthesis of peptide or non-peptide compounds (mimetics) which would
be useful for the same diagnostic and therapeutic applications as the
antibodies (e.g. Saragovi et al., (1991) Science 253:792-795).
When the humanized LK26 specific antibodies of the invention are used in
vitro, the antibodies are typically administered in a composition
comprising a pharmaceutical carrier. A pharmaceutical carrier can be any
compatible, non-toxic substance suitable for delivery of the monoclonal
antibodies to the patient, Sterile water, alcohol, fats, waxes, and inert
solids may be included in the carrier. Pharmaceutically accepted adjuvants
(buffering agents, dispersing agent) may also be incorporated into the
pharmaceutical composition.
The humanized antibodies compositions of the invention may be administered
to a patient in a variety of ways. Preferably, the pharmaceutical
compositions may be administered parenterally, i.e., subcutaneously,
intramuscularly or intravenously. Thus, this invention provides
compositions for parenteral administration which comprise a solution of
the human monoclonal antibody or a cocktail thereof dissolved in an
acceptable carrier, preferably an aqueous carrier. A variety of aqueous
carriers can be used, e.g., water, buffered water, 0.4% saline, 0.3%
glycine and the like. These solutions are sterile and generally free of
particulate matter. These compositions may be sterilized by conventional,
well known sterilization techniques. The compositions may contain
pharmaceutically acceptable auxiliary substances as required to
approximate physiological conditions such as pH adjusting and buffering
agents, toxicity adjusting agents and the like, for example sodium
acetate, sodium chloride, potassium chloride, calcium chloride, sodium
lactate, etc. The concentration of antibody in these formulations can vary
widely, e.g., from less than about 0.5%, usually at or at least about 1%
to as much as 15 or 20% by weight and will be selected primarily based on
fluid volumes, viscosities, etc., in accordance with the particular mode
of administration selected.
Actual methods for preparing parenterally administrable compositions and
adjustments necessary for administration to subjects will be known or
apparent to those skilled in the art and are described in more detail in,
for example, Remington's Pharmaceutical Science, 15th Ed., Mack Publishing
Company, Easton, Pa. (1980), which is incorporated herein by reference.
The subject invention provide numerous humanized antibodies specific for
the LK26 antigen based on the discovery that the CDR regions of the murine
monoclonal antibody could be spliced into a human acceptor framework so as
to produce a humanized recombinant antibody specific for the LK26 antigen.
Preferred humanized LK26 specific antibodies contain additional change in
the framework region (or in other regions) to increasing binding for LK26
antigen. Particularly preferred embodiments of the invention are the
exemplified humanized antibody molecules having superior binding
properties for LK26.
The following examples are offered by way of illustration of the invention,
and should not be interpreted as a limitation of the invention.
5.1. EXAMPLES
In the following examples all necessary restriction and modification
enzymes, plasmids and other reagents and materials were obtained from
commercial sources unless otherwise indicated.
Unless otherwise indicated, all general recombinant DNA methodology was
performed as described in "Molecular Cloning, A Laboratory Manual" (1989)
Eds J. Sambrook et al., published by Cold spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y.
In the following examples these abbreviations may be employed:
______________________________________
dCTP deoxycytidine triphosphate
dATP deoxyadenosine triphosphate
dGTP deoxyguanosine triphosphate
dTTP deoxythymidine triphosphate
DTT dithiothreitol
C cytosine
A adenine
G guanine
T thymine
PBS phosphate buffered saline
PBSB phosphate buffered saline
containing 0.5% (w/v) bovine serum
albumin
PBST phosphate buffered saline
containing 0.05% (v/v) Tween -20
______________________________________
5.1.1. Example 1
Production of Humanized Antibodies Specific for the LK26 Antigen
The source of the donor CDRs used to prepare these recombinant antibodies
was a murine monoclonal antibody, mAbLK26, which is specific for the LK26
antigen of certain human cancers. The LK26 monoclonal antibody was
produced by immunization of (BALB/c.times.C57BL/6) F.sub.1 mice with
LU-75(c) choriocarcinoma cell lines and subsequent production and
screening of hybridoma cells. Cytoplasmic RNA was prepared from the mAb
LK26 hybridoma cell line by the method of Favoloro, J. et al., (1980),
Methods in Enzymology 65:718-749). cDNA was synthesized using Ig variable
region primers as follows: for the Ig heavy chain variable region (VH),
the primer CG2aFOR (5' GGAAGCTTAGACCGATGGGGCTGTTGTTTTG 3') (SEQ ID NO: 1);
for the light chain variable region (VK), the primer CK2FOR (5'
GGAAGCTTGAAGATGGATACAGTTGGTGCAGC 3')(SEQ ID NO: 2). cDNA synthesis
reactions consisted of 5 .mu.g RNA, 20 pmol CG2aFOR or CK2FOR, 250 .mu.M
each of dATP, dCTP, dGTP and dTTP, 100 mM TrisHCl pH8.3, 140 mM KCl, 10 mM
DTT, 10 mM MgCl.sub.2 and 31.5 units of RNase inhibitor (Pharmacia, Milton
Keynes, U.K.) in a total volume of 50 .mu.l. Samples were heated to
70.degree. C. for 10 minutes (min) then slowly cooled to 42.degree. C.
over a period of 30 min. 100 units of Moloney Murine Leukaemia virus
(MMLV) reverse transcriptase (Life Technologies Ltd, Paisley, U.K.) was
added and incubation at 42.degree. C. continued for 1 hour.
VH and VK cDNAs were then amplified using the polymerase chain reaction
(PCR) as described by Saiki, R. K. et al., (1988), Science 239:487-491.
The primers used were:
(SEQ ID NO:1)
CG2aFOR (5'GGAAGCTTAGACCGATGGGGCTGTTGTTTTG 3')
(SEQ ID NO:2)
CK2FOR (5' GGAAGCTTGAAGATGGATACAGTTGGTGCAGC 3')
(SEQ ID NO:3)
VH1BACK (5' AGGTSMARCTGCAGSAGTCWGG 3')
(SEQ ID NO;4)
VK4BACK (5' GACATTGAGCTCACCCAGTCTCCA 3')
where M=C or A, S=C or G, R=A or G and W=A or T. Such primers and their use
in the PCR amplification of mouse Ig DNA are described by Orlandi, R. et
al., (1989), Proc. Natl Acad. Sci. USA, 86:3833-3837. For PCR
amplification of VH, 5 .mu.l RNA/cDNA hybrid was mixed with 25 pmol
CG2aFOR and VH1BACK primers. For PCR amplification of VK, 5 .mu.l RNA/cDNA
hybrid was mixed with 25 pmol CK2FOR and VK4BACK primers. To these
mixtures was added 200 .mu.M each of dATP, dCTP, dGTP and dTTP, 67 mM
TrisHCl pH8.8, 17 mM (NH.sub.4).sub.2 SO.sub.4, 10 mM MgCl.sub.2, 0.02%
(w/v) gelatin and 2.5 units of AmpliTaq DNA polymerase (Perkin Elmer Ltd,
Beaconsfield, U.K.) in a total volume of 50 .mu.l. These were then
subjected to 25 thermal cycles of PCR at 94.degree. C., 30 s; 50.degree.
C., 40 s; 72.degree. C., 30 s; ending with 5 min at 72.degree. C. For
cloning and sequencing, amplified DNA was purified by electrophoresis in a
low melting point agarose gel and by Elutip-d column chromatography
(Schleicher and Schuell, Dussel, Germany). Amplified VH DNA was cut with
HindIII and PstI and cloned into M13mp18 or M13mp19 cut with HindIII and
PstI (Life Technologies Ltd, Paisley, U.K.). Amplified VK DNA was cut with
HindIII and SacI and cloned into HindIII and SacI cut M13mp18 or M13mp19
(Life Technologies Ltd, Paisley, U.K,).
The resulting clones were sequenced by the dideoxy method (Sanger, F. et
al., (1977), Proc. Natl Acad. Sci. USA 74:5463-5467) using Sequenase
(United States Biochemical, Cleveland, Ohio, USA). The DNA and protein
sequences of the LK26 VH and VK domains are shown in FIGS. 1 and 2. The
location of the CDRs was determined with reference to Kabat, E. A. et al.,
(1987) "Sequences of Protein of Immunological Interest", US Department of
Health and Human Services, US Government Printing Office, and utilizing
computer assisted alignment with other VH and VK sequences.
The transfer of the murine CDRs to human frameworks was achieved by
oligonucleotide site-directed mutagenesis, based on the method of Nakamye,
K. and Eckstein, F. (1986) Nucleic Acids Res. 14:9679-9698. The human
framework regions chosen to receive the transplanted CDRs were NEWM or KOL
and REI for the heavy and light chains respectively. The structures of
these proteins have been solved crystallographically. The templates for
mutagenesis were human framework region genes containing irrelevant CDRs
and consisted of synthetic DNAs cloned into M13 phage (Riechmann, L. et
al., (1988) Nature 332:323-327).
The oligonucleotides used were:
NEWM VH:
VHCDR1
5'TGGCTGTCTCACCCAAGACAAGCCATAGCCGCTGAAGGTG
(SEQ ID NO:5)
AAGCCAGACGCGGTGCAGGTCAGGCT 3'
VHCDR2
5' GTTCTTGCTGGTGTCTCTCAGCATTGTCACTCTCCCCTTCAC
(SEQ ID NO:6)
ACTGTCTGCATAGTAGGTATAACTACCACCACTACTAATCATTG
CAACCCACTCAAGACC 3'
VHCDR3
5'TGAGGAGACGGTGACCAGGCTCCCTTGGCCCCAGTAAGCAA
(SEQ ID NO:7)
ACCAGGCGGGATCGTCCCCATGTCTTGCACAATAATA 3'
KOL VH:
VHCDR1
5'CCTGTCTCACCCAAGACAACCCATAGCCGCTGAAGGTGAAGC
(SEQ ID NO:8)
CAGATGCGGAGCAGGACAGGC 3'
VHCDR2
5'GAACAATGTGTTCTTGGCGTTGTCTCGCGATATTGCAAATCT
(SEQ ID NO:9)
ACCCTTCACACTGTCTGCATAGTAGGTATAACTACCACCACTAC
TAATCATTGCAACCCACTCAAGACCTTTTCC 3'
VHCDR3
5' CCAATAAGCAAACCAGGCGGGATCGTCCCCATGTCTTGCA
(SEQ ID NO:10)
CAAAAATAGAC 3'
RET VK:
VKCDR1
5' CTTCTGCTGGTACCAGTGCAAGTTGTTGGAACTTATACTT
(SEQ ID NO:11)
GAGCTGACACTACAGGTGATGGTCAC 3'
VKCDR2
5' TCTGCTTGGCACACCAGAAGCCAGGTTGGATGTGCCGTA
(SEQ ID NO:12)
GATCAGCAGCTT 3'
VKCDR3
5'GGTCCCTTGGCCGAACGTGTACATGTACGGGTAACTACT
(SEQ ID NO:13)
CCACTGTTGGCAGTAGTAGGTGGC 3'
A number of additional, murine residues were introduced into the variable
region frameworks by extension of the CDR primers. Specifically:
______________________________________
NEWM V(24) changed to A
(NEWM VHCDR1
oligonucleotide)
NEWM S(27) changed to F
(NEWM VHCDR1
oligonucleotide)
NEWM I(48) changed to V
(NEWM VHCDR2
oligonucleotide)
NEWM G(49) changed to A
(NEWM VHCDR2
oligonucleotide)
NEWM V(71) changed to R
(NEWM VHCDR2
oligonucleotide)
KOL S(24) changed to A
(KOL VHCDR1
oligonucleotide)
KOL I(28) changed to T
(KOL VHCDR1
oligonucleotide)
KOL T(68) changed to A
(KOL VHCDR2
oligonucleotide)
KOL S(74) changed to A
(KOL VHCDR2
oligonucleotide)
______________________________________
These residues that have been changed are believed to be important for
retaining original antigen specificity. Although the invention is not
dependent upon any particular explanation for the results obtained by
making the additional residue changes, some possible explanations for
their significance are as follows:
The change of residues NEWM V(24) and KOL S(24) to the smaller A
facilitates the accommodation of the heterologous CDR1 loop. The NEWM
S(27) to F change was made because S(27) is an unusual residue in subgroup
II human heavy chains (Riechmann et al., (1988) Nature 332:323-327). Amino
acids VH(27-30, 47-49, 71) are residues of the `vernier zones` as defined
by Foote and Winter (Foote, J. and Winter G. (1992) J. Mol. Biol.
224:487-499. These zones are important for adjusting CDR structures to
promote antigen binding. This explanation accounts for the changes NEWM
S(27) to F, NEWM I(48) to V, NEWM G(49) to A, NEWM V(71) to R and KOL
I(28) to T.
For site directed mutagenesis the VH and VK oligonucleotides encoding the
murine CDRs were phosphorylated with T4 Kinase (Life Technologies Ltd,
Paisley, U.K.). A 25 fold molar excess of each of the three VH or VK
primers were added to 0.5 .mu.g of appropriate VH or VK single stranded
template DNA in M13 (NEWM VH: M13VHPCR1; KOL VH M13MN14VH; REI: M13VKPCR2)
in 40 mM Tris HCl pH7.5, 20 mM MgCl.sub.2, 50 mM NaCl and annealed by
heating to 90.degree. C. for a few minutes and slowly cooling to
37.degree. C. The annealed DNA was extended with 2.5 units of T7 DNA
polymerase (cloned, United States Biochemical, Cleveland, Ohio, USA) in a
reaction mixture containing 0.5 units of T4 DNA ligase (Life Technologies
Ltd, Paisley, U.K.), 0.25 mM of each of dATP, dGTP, dTTP, and dCTP
(Pharmacia, Milton Keynes, U.K.), 40 mM Tris HCl pH7.5, 20 mM MgCl.sub.2,
50 mM NaCl, 6.5 mM DTT and 1 mM ATP in a total volume of 30 .mu.l. The
mixture was incubated at room temperature for 1 h. A 1 .mu.l aliquot of
this extension/ligation mixture was then used in an asymmetric PCR for the
specific amplification of the newly synthesized strand. The reaction
contained 1 .mu.l extension/ligation mixture, 250 .mu.M of each of dATP,
dGTP, dTTP and dCTP, 67 mM Tris HCl pH8.8, 17 mM (NH.sub.4) .sub.2
SO.sub.4, 10 mM MgCl.sub.2, 0.02% (w/v) gelatin, 2.5 units of AmpliTaq DNA
polymerase and 25 pmol of appropriate oligonucleotide primer (5'
AACAGCTATGACCATG 3' (SEQ ID NO:14) for NEWM VH and KOL VH; 5'
CTCTCTCAGGGCCAGGCGGTGA 3' (SEQ ID NO:15) for REI VK) in a total volume of
50 .mu.l. The reaction mixtures were subjected to 30 thermal cycles of PCR
at 94.degree. C., 30 s; 55.degree. C., 30 s; 72.degree. C., 1 min ending
with 72.degree. C., 5 min. The newly synthesized strand was then amplified
by adding 20 pmol of appropriate oligonucleotide primer (5'
GTAAAACGACGGCCAGT 3' (SEQ ID NO:16) for NEWM VH and KOL VH and 5'
GCGGGCCTCTTCGCTATTACGC 3' (SEQ ID NO:17) for REI VK) and adjusting the
reaction mixture to include a further 5 n moles of each of dATP, dGTP,
dTTP and dCTP and 2.5 Units of AmpliTaq. The reactions were subjected to a
further 20 PCR cycles as above. The amplified VH and VK DNAs were purified
from 1.5% w/v low melting point agarose gels by elutip-d column
chromatography. Purified DNA was digested with HindIII and BamHI plus RsaI
(for VHs) or BstXI (for VKs) (all restriction enzymes were obtained from
Life Technologies Ltd, Paisley, U.K.). There is an RsaI site in the
parental VHPCR1 and MN14VH and a BstXI site in the parental VKPCR2 but
these sites are deleted during mutagenesis. These digestions therefore
select for newly synthesized DNA. The HindIII/BamHI digested VH and VK
DNAs were ligated into HindIII/BamHI cut M13mp18 or M13mp19 (both from
Pharmacia, Milton Keynes, U.K.) and transformed into competent E. coli TG1
(Amersham International plc, Amersham, U.K.). Single stranded DNA was
prepared from individual `plaques` and sequenced by the dideoxy method
using Sequenase (United States Biochemical, Cleveland, Ohio, USA)
according to Manufacturer's instructions. Triple CDR mutants were
identified in this way and selected for construction of VH and VK
expression vectors.
The expression vectors for the humanized VH and VK genes, pSVgpt and pSVhyg
are shown in FIGS. 3 and 4. The humanized VH genes, together with the
immunoglobulin heavy chain promoter, appropriate splice sites and signal
peptide sequences were excised from the M13 clones with HindIII and BamHI
and ligated into the heavy chain expression vector, pSVgpt. This vector
contains the murine heavy chain immunoglobulin enhancer, the gpt gene
under control of the SV40 promoter/enhancer for selection in mammalian
cells, the human IgG1 constant region domain and sequences for replication
and selection in E. coli. The humanized VK gene was cloned into the light
chain expression vector pSVhyg in the same way. All features of pSVhyg are
the same as in pSVgpt except that the gpt gene is replaced by the gene for
hygromycin resistance (hyg) and a human kappa constant region is included
instead of the IgG1 constant region.
For transfection into mammalian cells 10 .mu.g of the heavy chain
expression vector DNA and 20 .mu.g of the light chain vector DNA were
linearized by digestion with PvuI (Life Technologies Ltd, Paisley, U.K.),
coprecipitated with ethanol and redissolved in 20 .mu.l of water. The
recipient cell line was NSO, a non-immunoglobulin producing mouse myeloma,
obtained from the European collection of Animal Cell Cultures, Porton,
U.K., ECAC No. 85110505 cells were grown in Dulbecco's Modified Eagle's
Medium supplemented with 10% foetal calf serum and antibiotics (DMEM)
(Life Technologies Ltd, Paisley, U.K.). Approximately 10.sup.7 NSO cells
were harvested by centrifugation and resuspended in 0.5 ml DMEM, the
digested DNA was added and the cells transferred to a cuvette and placed
on ice for 5 min. A single pulse of 170 volts, 960.mu. farads was
administered (Genepulser, BioRad, Richmond, Calif., U.S.A.). After a
further 30 min on ice the cells were replaced in a flask in 20 ml DMEM and
allowed to recover for 24 hours. After this time the cells were
distributed into a 24 well plate in selective medium (DMEM with 0.8
.mu.g/ml mycophenolic acid and 250 .mu.g/ml xanthine). After 3 to 4 days
the medium was changed for fresh selective medium. Colonies of transfected
cells were visible after 10 to 14 days.
The production of human antibody in the wells containing transfected clones
was measured by ELISA. Capture antibody, goat anti-human IgG, gamma chain
specific (Sera-Lab Ltd, Crawley Down, U.K.) was diluted to 5 .mu.g/ml in
50 mM carbonate buffer pH9.6, and used to coat polystyrene ELISA plates
(Dynatech Immulon 1), 200 .mu.l per well, overnight at 4.degree. C. After
washing 3 times with PBST, 50-100 .mu.l of the culture medium to be
screened was added to the wells and incubated at 37.degree. C. for 60 min.
The wells were washed again with PBST and the reporter antibody,
peroxidase-conjugated goat anti-human IgG, gamma chain specific (Sera-Lab
Ltd, Crawley Down, U.K.) or peroxidase-conjugated goat anti-human kappa
chain (Sera-Lab Ltd, Crawley Down, U.K) was added at 100 ng per well and
the plate incubated for a further 60 min. The plate was washed as before
then the colour was developed. Substrate buffer was prepared by mixing 100
mM citric acid and 100 mM disodium hydrogen phosphate to pH5.0. 25 mg of
o-phenylenediamine was dissolved in 50 ml and 5 .mu.l of 30% hydrogen
peroxide added just before use. 200 .mu.l was dispensed per well and
incubated at room temperature in the dark. The reaction was stopped by
addition of 50 .mu.l per well of 12.5% sulphuric acid and the absorbances
were read at 492 nm.
Positive cell clones were expanded for antibody purification. For the final
expansion to production volume the cells were diluted in DMEM containing
10% IgG-free fetal calf serum. For small scale purification 500 ml of
conditioned medium from static flask or spinner cultures was harvested by
centrifugation. 0.1 volumes of 1.0 M TrisHCl pH8.0 and 0.5 to 1.0 ml of
Protein A-agarose (Boehringer Mannheim, Lewes, U.K.) were added. This was
stirred overnight at room temperature then collected on a disposable
column. This was washed with 10 column volumes of 0.1 M TrisHCl pH8.0, 10
column volumes of 0.01M TrisHCl pH8.0 and eluted with 0.1 M glycine
buffer, pH3.0. 1.0 ml fractions were collected into tubes containing 100
.mu.l of 1.0 M TrisHCl, pH8.0. Fractions containing antibody were pooled
and dialysed against PBS. The concentrations of the antibody preparations
were determined using a Micro BCA Protein Assay Reagent Kit (Pierce,
Rockford, USA). Samples were checked by running on 10% SDS-polyacrylamide
gels.
The chimeric LK26 antibody, in which the murine constant region domains of
the heavy and light chains had been replaced by the human constant regions
used in the humanized antibody, was constructed as described by Orlandi et
al., (1989). Three hybrid chimeric/humanized antibodies were constructed
consisting of the chimeric heavy chain with the humanized light chain and
the humanized heavy chain (from NEWM and KOL mutagenesis) with the
chimeric light chain.
None of these hybrid antibodies showed binding to the SW620 target cells
equivalent to the chimeric antibody. This indicates that further framework
changes, in both the VH and VK chains, are necessary to restore antigen
binding.
Four further versions of the LK26HuVH and two further versions of the
LK26HuVK were designed. The amino acid sequences of these VHs and VKs are
shown in Table 1.
Table 1 shows the variable region sequences of LK26HuVH (SEQ ID NO: 18),
LK26HuVHFAIS,N (SEQ ID NO: 19), LK26HuVH SLF (SEQ ID NO: 21), LK26HuVHI,I
(SEQ ID NO: 20), LK26KOLHuVH (SEQ ID NO: 22), LK26HuVK (SEQ ID NO: 23),
LK26HuVKY (SEQ ID NO: 24) and LK26HuVKPW,Y (SEQ ID NO: 25 with the proviso
that amino acid 72 is not Phe but is Tyr). Murine framework residues are
shown in lower case. Some framework residues in NEWM and REI are unusual
for human subgroup II heavy chains or human subgroup I kappa chains,
respectively, these have been replaced by the residues commonly found at
these positions and are underlined in the table.
The additional changes to the HuVH and HuVK constructs are shown below
(numbering according to Kabat et al., ibid):
LK26HuVHFAIS,N (68-71, 74)
LK26HuVHSLF (78-80)
LK26HuVHI,I (93,95)
LK26HuVKY (72)
LK26HuVKPW,Y (47-48, 72)
These new versions were constructed by mutagenesis of the original reshaped
heavy and light chain M13 single stranded DNA clones. The method of
Higuchi, R. et al. (1988) Nucleic Acids Res. 16:7351-7367, which utilizes
overlapping PCR amplification with mutagenic primers, was employed. The
modified variable regions were cloned into the expression vector pSVgpt or
pSVhyg as before and cotransfected with either the MuVK or MuVH plasmids
into NSO cells. Antibody producing cell clones were selected, expanded and
purified for testing. Subsequent to this, fully humanized version
antibodies consisting of the modified HuVHs and HuVKs were prepared in the
same way.
Another version of the humanized light chain produced was LK26HuVKPW (SEQ
ID NO: 25). LK26HuVKPW (SEQ ID NO: 25) is similar to LK26HuVKPW,Y (SEQ ID
NO: 25, with the proviso that amino acid 72 is not Phe but is Tyr), but
lacks the change at position 71.
5.1.2. Example 2
Specific Binding of Humanized LK26 Antibodies to Carcinoma Cells
The recombinant antibodies have been tested in ELISAs using the SW620
target cells. The ELISA method used is as follows:
SW620 cells are diluted to 1.5.times.10.sup.5 -2.5.times.10.sup.5 cells/ml
in DMEM, 10% FCS and 200l (ie 3-5.times.10.sup.4 cells) added to each
well. Cells are grown until nearly confluent (about 2 days). Plates are
washed 2.times. with PBS and 100 .mu.l antibody (diluted in DMEM) added.
Incubation is carried out at 4.degree. C. for 1 hour. The wells are washed
3.times. with PBS and 100 .mu.l of appropriate reporter antibody added, ie
either goat anti-human IgG1, HRPO conjugate (Sera-lab, 0.4 mg/ml, diluted
1:500 in DMEM) or goat anti-mouse IgG1, HRPO conjugate (Sera-lab, 0.4
mg/ml, diluted 1:500 in DMEM), incubation is carried out at 4.degree. C.
for 1 hour. Wells are washed 3.times. with PBS and bound reporter antibody
detected using H.sub.2 O.sub.2 and o-phenylenediaminedihydrochloride and
the OD 492 nm measured.
The humanized antibodies tested in ELISAs are LK26HuVHSLF (SEQ ID NO:
21)/HuVKPW,Y (SEQ ID NO: 25 with the proviso that amino acid 72 is not Phe
but is Tyr), LK26HuVHFAIS,N (SEQ ID NO: 19)/HuVKPW,Y (SEQ ID NO: 25 with
the proviso that amino acid 72 is not Phe but is Tyr) and LK26KOLHUVH (SEQ
ID NO: 22)/HuVKPW,Y (SEQ ID NO: 25 with the proviso that amino acid 72 is
not Phe but is Tyr). The data are presented graphically below. The test
data of the various recombinant antibodies indicate that of particular
value for the restoration of antigen binding is the inclusion of the P and
W residues into the humanized light chain. Furthermore, the data show that
these inclusions facilitate the proper association of the VH and VL
domains. This invention therefore also relates to the inclusion into the
humanized antibody of these and other VH and VL residues to facilitate
proper VH and VL association and thereby antigen binding.
The test data indicates that these humanized antibodies retain the binding
properties of the original murine and chimeric antibodies. In particular
the LK26HuVHFAIS,N (SEQ ID NO: 19)/HuVKPW,Y (SEQ ID NO: 25 with the
proviso that amino acid 72 is not Phe but is Tyr) and the LK26KOLHuVH (SEQ
ID NO: 22)/HuVKPW,Y (SEQ ID NO: 25 with the proviso that amino acid 72 is
not Phe but is Tyr) exhibit binding affinities higher than the chimeric
antibody. Such recombinant antibodies (of which these are examples)
therefore provide for novel, recombinant antibody molecules for the
diagnosis and therapy of human cancers characterized by the expression of
the LK26 antigen.
TABLE 1
__________________________________________________________________________
LK26HuVH: QVQLQESGPGLVRPSQTLSLTCTaSGfTFSGYGLSWVRQ
(SEQ ID NO:18)
PPGRGLEWvaMISSGGSYTYYADSVKGRVTMLrDTSKNQ
FSLRLSSVTAADTAVYYCARHGDDPAWFAYWGQGSLVTV
SS
LK26HUVHFAIS,N:
QVQLQESGPGLVRPSQTLSLTCTaSGfTFSGYGL
(SEQ ID NO:19)
SWVRQPPGRGLEWvaMISSGGSYTYYADSVKGRf
aisrDnSKNQFSLRLSSVTAADTAVYYCARHGDD
PAWFAYWGQGSLVTVSS
LK26HuVHI,I:
QVQLQESGPGLVRPSQTLSLTCTaSGfTFSGYGLSWVRQ
(SEQ ID NO:20)
PPGRGLEWvaMISSGGSYTYYADSVKGRVTMLrDTSKNQ
FSLRLSSVTAADTAiYiCARHGDDPAWFAYWGQGSLVTV
SS
LK26HuVHSLF:
QVQLQESGPGLVRPSQTLSLTCTaSGfTFSGYGLSWVRQ
(SEQ ID NO:21)
PPGRGLEWVaMISSGGSYTYYADSVKGRVTMLrDTSKNs
lfLRLSSVTAADTAVYYCARHGDDPAWFAYWGQGTTVTV
SS
LK26KOLHuVH:
EVQLVESGGGVVQPGRSLRLSCSaSGFtFSGYGLSWVRQ
(SEQ ID NO:22)
APGKGLEWVAMISSGGSYTYYADSVKGRFaISRDNaKNT
LFLQMDSLRPEDTGVYFCARHGDDPAWFAYWGQGTPVTV
SS
LK26HuVK: DIQLTQSPSSLSASVGDRVTITCSVSSSISSNNLHWYQQ
(SEQ ID NO:23)
KPGKAPKLLIYGTSNLASGVPSRFSGSGSGTDFTFTISS
LQPEDIATYYCQQWSSYPYMYTFGQGTKVEIK
LK26HuVKY:
DIQbTQSPSSLSASVGDRVTITCSVSSSISSNNLHWYQQ
(SEQ ID NO:24)
KPGKAPKLLIYGTSNLASGVPSRFSGSGSGTDYTFTISS
LQPEDIATYYCQQWSSYPYMYTFGQGTKVEIK
LK26HuVKPW1,Y:
DIQLTQSPSSLSASVGDRVTITCSVSSSISSNNLHWYQQ
(SEQ ID NO:25
KPGKAPKpWIYGTSNLASGVPSRFSGSGSGTDFTFTISS
with the proviso
LQPEDIATYYCQQWSSYPYMYTFGQGTKVEIK
that amino acid
72 is not Phe
but is
__________________________________________________________________________
Tyr)
Incorporation by Reference
All patents, patents applications, and publications cited are incorporated
herein by reference.
Equivalents
The foregoing written specification is considered to be sufficient to
enable one skilled in the art to practice the invention. Indeed, various
modifications of the above-described makes for carrying out the invention
which are obvious to those skilled in the field of molecular biology or
related fields are intended to be within the scope of the following
claims.
__________________________________________________________________________
# SEQUENCE LISTING
- (1) GENERAL INFORMATION:
- (iii) NUMBER OF SEQUENCES: 31
- (2) INFORMATION FOR SEQ ID NO: 1:
- (i) SEQUENCE CHARACTERISTICS:
#pairs (A) LENGTH: 31 base
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# 31 GGGG CTGTTGTTTT G
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# 32 ATAC AGTTGGTGCA GC
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- TGGCTGTCTC ACCCAAGACA AGCCATAGCC GCTGAAGGTG AAGCCAGACG CG - #GTGCAGGT
60
# 66
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- GTTCTTGCTG GTGTCTCTCA GCATTGTCAC TCTCCCCTTC ACACTGTCTG CA - #TAGTAGGT
60
# 102 CTAA TCATTGCAAC CCACTCAAGA CC
- (2) INFORMATION FOR SEQ ID NO: 7:
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- TGAGGAGACG GTGACCAGGC TCCCTTGGCC CCAGTAAGCA AACCAGGCGG GA - #TCGTCCCC
60
# 78 TA
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#8: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:
- CCTGTCTCAC CCAAGACAAC CCATAGCCGC TGAAGGTGAA GCCAGATGCG GA - #GCAGGACA
60
# 63
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#9: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:
- GAACAATGTG TTCTTGGCGT TGTCTCGCGA TATTGCAAAT CTACCCTTCA CA - #CTGTCTGC
60
- ATAGTAGGTA TAACTACCAC CACTACTAAT CATTGCAACC CACTCAAGAC CT - #TTTCC
117
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# 51CAGGCGG GATCGTCCCC ATGTCTTGCA CAAAAATAGA C
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- CTTCTGCTGG TACCAGTGCA AGTTGTTGGA ACTTATACTT GAGCTGACAC TA - #CAGGTGAT
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#12: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:
# 51CCAGAAG CCAGGTTGGA TGTGCCGTAG ATCAGCAGCT T
- (2) INFORMATION FOR SEQ ID NO: 13:
- (i) SEQUENCE CHARACTERISTICS:
#pairs (A) LENGTH: 63 base
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unkn - #own
(D) TOPOLOGY: unknown
- (ii) MOLECULE TYPE: DNA (genomic)
#13: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:
- GGTCCCTTGG CCGAACGTGT ACATGTACGG GTAACTACTC CACTGTTGGC AG - #TAGTAGGT
60
# 63
- (2) INFORMATION FOR SEQ ID NO: 14:
- (i) SEQUENCE CHARACTERISTICS:
#pairs (A) LENGTH: 16 base
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unkn - #own
(D) TOPOLOGY: unknown
- (ii) MOLECULE TYPE: DNA (genomic)
#14: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:
# 16
- (2) INFORMATION FOR SEQ ID NO: 15:
- (i) SEQUENCE CHARACTERISTICS:
#pairs (A) LENGTH: 22 base
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unkn - #own
(D) TOPOLOGY: unknown
- (ii) MOLECULE TYPE: DNA (genomic)
#15: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:
# 22GGT GA
- (2) INFORMATION FOR SEQ ID NO: 16:
- (i) SEQUENCE CHARACTERISTICS:
#pairs (A) LENGTH: 17 base
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unkn - #own
(D) TOPOLOGY: unknown
- (ii) MOLECULE TYPE: DNA (genomic)
#16: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:
# 17 T
- (2) INFORMATION FOR SEQ ID NO: 17:
- (i) SEQUENCE CHARACTERISTICS:
#pairs (A) LENGTH: 22 base
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unkn - #own
(D) TOPOLOGY: unknown
- (ii) MOLECULE TYPE: DNA (genomic)
#17: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:
# 22TAC GC
- (2) INFORMATION FOR SEQ ID NO: 18:
- (i) SEQUENCE CHARACTERISTICS:
#acids (A) LENGTH: 119 amino
(B) TYPE: amino acid
(C) STRANDEDNESS: unkn - #own
(D) TOPOLOGY: unknown
- (ii) MOLECULE TYPE: protein
#18: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:
- Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Le - #u Val Arg Pro Ser Gln
# 15
- Thr Leu Ser Leu Thr Cys Thr Ala Ser Gly Ph - #e Thr Phe Ser Gly Tyr
# 30
- Gly Leu Ser Trp Val Arg Gln Pro Pro Gly Ar - #g Gly Leu Glu Trp Val
# 45
- Ala Met Ile Ser Ser Gly Gly Ser Tyr Thr Ty - #r Tyr Ala Asp Ser Val
# 60
- Lys Gly Arg Val Thr Met Leu Arg Asp Thr Se - #r Lys Asn Gln Phe Ser
#80
- Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Th - #r Ala Val Tyr Tyr Cys
# 95
- Ala Arg His Gly Asp Asp Pro Ala Trp Phe Al - #a Tyr Trp Gly Gln Gly
# 110
- Ser Leu Val Thr Val Ser Ser
115
- (2) INFORMATION FOR SEQ ID NO: 19:
- (i) SEQUENCE CHARACTERISTICS:
#acids (A) LENGTH: 119 amino
(B) TYPE: amino acid
(C) STRANDEDNESS: unkn - #own
(D) TOPOLOGY: unknown
- (ii) MOLECULE TYPE: protein
#19: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:
- Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Le - #u Val Arg Pro Ser Gln
# 15
- Thr Leu Ser Leu Thr Cys Thr Ala Ser Gly Ph - #e Thr Phe Ser Gly Tyr
# 30
- Gly Leu Ser Trp Val Arg Gln Pro Pro Gly Ar - #g Gly Leu Glu Trp Val
# 45
- Ala Met Ile Ser Ser Gly Gly Ser Tyr Thr Ty - #r Tyr Ala Asp Ser Val
# 60
- Lys Gly Arg Phe Ala Ile Ser Arg Asp Asn Se - #r Lys Asn Gln Phe Ser
#80
- Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Th - #r Ala Val Tyr Tyr Cys
# 95
- Ala Arg His Gly Asp Asp Pro Ala Trp Phe Al - #a Tyr Trp Gly Gln Gly
# 110
- Ser Leu Val Thr Val Ser Ser
115
- (2) INFORMATION FOR SEQ ID NO: 20:
- (i) SEQUENCE CHARACTERISTICS:
#acids (A) LENGTH: 119 amino
(B) TYPE: amino acid
(C) STRANDEDNESS: unkn - #own
(D) TOPOLOGY: unknown
- (ii) MOLECULE TYPE: protein
#20: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:
- Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Le - #u Val Arg Pro Ser Gln
# 15
- Thr Leu Ser Leu Thr Cys Thr Ala Ser Gly Ph - #e Thr Phe Ser Gly Tyr
# 30
- Gly Leu Ser Trp Val Arg Gln Pro Pro Gly Ar - #g Gly Leu Glu Trp Val
# 45
- Ala Met Ile Ser Ser Gly Gly Ser Tyr Thr Ty - #r Tyr Ala Asp Ser Val
# 60
- Lys Gly Arg Val Thr Met Leu Arg Asp Thr Se - #r Lys Asn Gln Phe Ser
#80
- Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Th - #r Ala Ile Tyr Ile Cys
# 95
- Ala Arg His Gly Asp Asp Pro Ala Trp Phe Al - #a Tyr Trp Gly Gln Gly
# 110
- Ser Leu Val Thr Val Ser Ser
115
- (2) INFORMATION FOR SEQ ID NO: 21:
- (i) SEQUENCE CHARACTERISTICS:
#acids (A) LENGTH: 119 amino
(B) TYPE: amino acid
(C) STRANDEDNESS: unkn - #own
(D) TOPOLOGY: unknown
- (ii) MOLECULE TYPE: protein
#21: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:
- Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Le - #u Val Arg Pro Ser Gln
# 15
- Thr Leu Ser Leu Thr Cys Thr Ala Ser Gly Ph - #e Thr Phe Ser Gly Tyr
# 30
- Gly Leu Ser Trp Val Arg Gln Pro Pro Gly Ar - #g Gly Leu Glu Trp Val
# 45
- Ala Met Ile Ser Ser Gly Gly Ser Tyr Thr Ty - #r Tyr Ala Asp Ser Val
# 60
- Lys Gly Arg Val Thr Met Leu Arg Asp Thr Se - #r Lys Asn Ser Leu Phe
#80
- Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Th - #r Ala Val Tyr Tyr Cys
# 95
- Ala Arg His Gly Asp Asp Pro Ala Trp Phe Al - #a Tyr Trp Gly Gln Gly
# 110
- Thr Thr Val Thr Val Ser Ser
115
- (2) INFORMATION FOR SEQ ID NO: 22:
- (i) SEQUENCE CHARACTERISTICS:
#acids (A) LENGTH: 119 amino
(B) TYPE: amino acid
(C) STRANDEDNESS: unkn - #own
(D) TOPOLOGY: unknown
- (ii) MOLECULE TYPE: protein
#22: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:
- Glu Val Gln Leu Val Glu Ser Gly Gly Gly Va - #l Val Gln Pro Gly Arg
# 15
- Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Ph - #e Thr Phe Ser Gly Tyr
# 30
- Gly Leu Ser Trp Val Arg Gln Ala Pro Gly Ly - #s Gly Leu Glu Trp Val
# 45
- Ala Met Ile Ser Ser Gly Gly Ser Tyr Thr Ty - #r Tyr Ala Asp Ser Val
# 60
- Lys Gly Arg Phe Ala Ile Ser Arg Asp Asn Al - #a Lys Asn Thr Leu Phe
#80
- Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Th - #r Gly Val Tyr Phe Cys
# 95
- Ala Arg His Gly Asp Asp Pro Ala Trp Phe Al - #a Tyr Trp Gly Gln Gly
# 110
- Thr Pro Val Thr Val Ser Ser
115
- (2) INFORMATION FOR SEQ ID NO: 23:
- (i) SEQUENCE CHARACTERISTICS:
#acids (A) LENGTH: 110 amino
(B) TYPE: amino acid
(C) STRANDEDNESS: unkn - #own
(D) TOPOLOGY: unknown
- (ii) MOLECULE TYPE: protein
#23: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:
- Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Le - #u Ser Ala Ser Val Gly
# 15
- Asp Arg Val Thr Ile Thr Cys Ser Val Ser Se - #r Ser Ile Ser Ser Asn
# 30
- Asn Leu His Trp Tyr Gln Gln Lys Pro Gly Ly - #s Ala Pro Lys Leu Leu
# 45
- Ile Tyr Gly Thr Ser Asn Leu Ala Ser Gly Va - #l Pro Ser Arg Phe Ser
# 60
- Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Th - #r Ile Ser Ser Leu Gln
#80
- Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gl - #n Trp Ser Ser Tyr Pro
# 95
- Tyr Met Tyr Thr Phe Gly Gln Gly Thr Lys Va - #l Glu Ile Lys
# 110
- (2) INFORMATION FOR SEQ ID NO: 24:
- (i) SEQUENCE CHARACTERISTICS:
#acids (A) LENGTH: 110 amino
(B) TYPE: amino acid
(C) STRANDEDNESS: unkn - #own
(D) TOPOLOGY: unknown
- (ii) MOLECULE TYPE: protein
#24: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:
- Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Le - #u Ser Ala Ser Val Gly
# 15
- Asp Arg Val Thr Ile Thr Cys Ser Val Ser Se - #r Ser Ile Ser Ser Asn
# 30
- Asn Leu His Trp Tyr Gln Gln Lys Pro Gly Ly - #s Ala Pro Lys Leu Leu
# 45
- Ile Tyr Gly Thr Ser Asn Leu Ala Ser Gly Va - #l Pro Ser Arg Phe Ser
# 60
- Gly Ser Gly Ser Gly Thr Asp Tyr Thr Phe Th - #r Ile Ser Ser Leu Gln
#80
- Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gl - #n Trp Ser Ser Tyr Pro
# 95
- Tyr Met Tyr Thr Phe Gly Gln Gly Thr Lys Va - #l Glu Ile Lys
# 110
- (2) INFORMATION FOR SEQ ID NO: 25:
- (i) SEQUENCE CHARACTERISTICS:
#acids (A) LENGTH: 110 amino
(B) TYPE: amino acid
(C) STRANDEDNESS: unkn - #own
(D) TOPOLOGY: unknown
- (ii) MOLECULE TYPE: protein
#25: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:
- Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Le - #u Ser Ala Ser Val Gly
# 15
- Asp Arg Val Thr Ile Thr Cys Ser Val Ser Se - #r Ser Ile Ser Ser Asn
# 30
- Asn Leu His Trp Tyr Gln Gln Lys Pro Gly Ly - #s Ala Pro Lys Pro Trp
# 45
- Ile Tyr Gly Thr Ser Asn Leu Ala Ser Gly Va - #l Pro Ser Arg Phe Ser
# 60
- Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Th - #r Ile Ser Ser Leu Gln
#80
- Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gl - #n Trp Ser Ser Tyr Pro
# 95
- Tyr Met Tyr Thr Phe Gly Gln Gly Thr Lys Va - #l Glu Ile Lys
# 110
- (2) INFORMATION FOR SEQ ID NO: 26:
- (i) SEQUENCE CHARACTERISTICS:
#pairs (A) LENGTH: 357 base
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unkn - #own
(D) TOPOLOGY: unknown
- (ii) MOLECULE TYPE: DNA (genomic)
#26: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:
- CAGGTSMARC TGCAGSAGTC WGGGGGAGAC TTGGTGAAGC CTGGAGGGTC CC - #TGAAACTC
60
- TCCTGTGCAG CCTCTGGATT CACTTTCAGT GGCTATGGCT TGTCTTGGGT TC - #GCCAGACT
120
- CCAGACAAGA GGCTGGAGTG GGTCGCAATG ATTAGTAGTG GTGGTAGTTA TA - #CCTACTAT
180
- GCAGACAGTG TGAAGGGGCG ATTCGCCATC TCCAGAGACA ATGCCAAGAA CT - #CCCTGTTC
240
- CTGCAAATGA GCAGTCTGAA GTCTGACGAC ACAGCCATTT ATATCTGTGC AA - #GACATGGG
300
- GACGATCCCG CCTGGTTTGC TTACTGGGGC CAAGGGACTC TAGTCACTGT CT - #CTGCT
357
- (2) INFORMATION FOR SEQ ID NO: 27:
- (i) SEQUENCE CHARACTERISTICS:
#pairs (A) LENGTH: 357 base
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unkn - #own
(D) TOPOLOGY: unknown
- (ii) MOLECULE TYPE: DNA (genomic)
#27: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:
- AGCAGAGACA GTGACTAGAG TCCCTTGGCC CCAGTAAGCA AACCAGGCGG GA - #TCGTCCCC
60
- ATGTCTTGCA CAGATATAAA TGGCTGTGTC GTCAGACTTC AGACTGCTCA TT - #TGCAGGAA
120
- CAGGGAGTTC TTGGCATTGT CTCTGGAGAT GGCGAATCGC CCCTTCACAC TG - #TCTGCATA
180
- GTAGGTATAA CTACCACCAC TACTAATCAT TGCGACCCAC TCCAGCCTCT TG - #TCTGGAGT
240
- CTGGCGAACC CAAGACAAGC CATAGCCACT GAAAGTGAAT CCAGAGGCTG CA - #CAGGAGAG
300
- TTTCAGGGAC CCTCCAGGCT TCACCAAGTC TCCCCCWGAC TSCTGCAGYT KS - #ACCTG
357
- (2) INFORMATION FOR SEQ ID NO: 28:
- (i) SEQUENCE CHARACTERISTICS:
#acids (A) LENGTH: 119 amino
(B) TYPE: amino acid
(C) STRANDEDNESS: unkn - #own
(D) TOPOLOGY: unknown
- (ii) MOLECULE TYPE: protein
#28: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:
- Gln Val Xaa Leu Gln Xaa Ser Gly Gly Asp Le - #u Val Lys Pro Gly Gly
# 15
- Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Ph - #e Thr Phe Ser Gly Tyr
# 30
- Gly Leu Ser Trp Val Arg Gln Thr Pro Asp Ly - #s Arg Leu Glu Trp Val
# 45
- Ala Met Ile Ser Ser Gly Gly Ser Tyr Thr Ty - #r Tyr Ala Asp Ser Val
# 60
- Lys Gly Arg Phe Ala Ile Ser Arg Asp Asn Al - #a Lys Asn Ser Leu Phe
#80
- Leu Gln Met Ser Ser Leu Lys Ser Asp Asp Th - #r Ala Ile Tyr Ile Cys
# 95
- Ala Arg His Gly Asp Asp Pro Ala Trp Phe Al - #a Tyr Trp Gly Gln Gly
# 110
- Thr Leu Val Thr Val Ser Ala
115
- (2) INFORMATION FOR SEQ ID NO: 29:
- (i) SEQUENCE CHARACTERISTICS:
#pairs (A) LENGTH: 330 base
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unkn - #own
(D) TOPOLOGY: unknown
- (ii) MOLECULE TYPE: DNA (genomic)
- (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..330
#29: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:
- GAC ATT GAG CTC ACC CAG TCT CCA GCA CTC AT - #G GCT GCA TCT CCA GGG
48
Asp Ile Glu Leu Thr Gln Ser Pro Ala Leu Me - #t Ala Ala Ser Pro Gly
# 15
- GAG AAG GTC ACC ATC ACC TGC AGT GTC AGC TC - #A AGT ATA AGT TCC AAC
96
Glu Lys Val Thr Ile Thr Cys Ser Val Ser Se - #r Ser Ile Ser Ser Asn
# 30
- AAC TTG CAC TGG TAC CAG CAG AAG TCA GAA AC - #C TCC CCC AAA CCC TGG
144
Asn Leu His Trp Tyr Gln Gln Lys Ser Glu Th - #r Ser Pro Lys Pro Trp
# 45
- ATT TAT GGC ACA TCC AAC CTG GCT TCT GGA GT - #C CCT CTT CGC TTC AGA
192
Ile Tyr Gly Thr Ser Asn Leu Ala Ser Gly Va - #l Pro Leu Arg Phe Arg
# 60
- GGC TTT GGA TCT GGG ACC TCT TAT TCT CTC AC - #A ATC AGC AGC ATG GAG
240
Gly Phe Gly Ser Gly Thr Ser Tyr Ser Leu Th - #r Ile Ser Ser Met Glu
# 80
- GCT GAA GAT GCT GCC ACT TAT TAC TGT CAA CA - #G TGG AGT AGT TAC CCG
288
Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gl - #n Trp Ser Ser Tyr Pro
# 95
- TAC ATG TAC ACG TTC GGA GGG GGG ACC AAG TT - #G GAA ATA AAA
# 330
Tyr Met Tyr Thr Phe Gly Gly Gly Thr Lys Le - #u Glu Ile Lys
# 110
- (2) INFORMATION FOR SEQ ID NO: 30:
- (i) SEQUENCE CHARACTERISTICS:
#acids (A) LENGTH: 110 amino
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
- (ii) MOLECULE TYPE: protein
#30: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:
- Asp Ile Glu Leu Thr Gln Ser Pro Ala Leu Me - #t Ala Ala Ser Pro Gly
# 15
- Glu Lys Val Thr Ile Thr Cys Ser Val Ser Se - #r Ser Ile Ser Ser Asn
# 30
- Asn Leu His Trp Tyr Gln Gln Lys Ser Glu Th - #r Ser Pro Lys Pro Trp
# 45
- Ile Tyr Gly Thr Ser Asn Leu Ala Ser Gly Va - #l Pro Leu Arg Phe Arg
# 60
- Gly Phe Gly Ser Gly Thr Ser Tyr Ser Leu Th - #r Ile Ser Ser Met Glu
# 80
- Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gl - #n Trp Ser Ser Tyr Pro
# 95
- Tyr Met Tyr Thr Phe Gly Gly Gly Thr Lys Le - #u Glu Ile Lys
# 110
- (2) INFORMATION FOR SEQ ID NO: 31:
- (i) SEQUENCE CHARACTERISTICS:
#pairs (A) LENGTH: 330 base
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unkn - #own
(D) TOPOLOGY: unknown
- (ii) MOLECULE TYPE: DNA (genomic)
#31: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:
- TTTTATTTCC AACTTGGTCC CCCCTCCGAA CGTGTACATG TACGGGTAAC TA - #CTCCACTG
60
- TTGACAGTAA TAAGTGGCAG CATCTTCAGC CTCCATGCTG CTGATTGTGA GA - #GAATAAGA
120
- GGTCCCAGAT CCAAAGCCTC TGAAGCGAAG AGGGACTCCA GAAGCCAGGT TG - #GATGTGCC
180
- ATAAATCCAG GGTTTGGGGG AGGTTTCTGA CTTCTGCTGG TACCAGTGCA AG - #TTGTTGGA
240
- ACTTATACTT GAGCTGACAC TGCAGGTGAT GGTGACCTTC TCCCCTGGAG AT - #GCAGCCAT
300
# 330 GTGA GCTCAATGTC
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